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The photodynamic deactivation of lysozyme in presence of acridine orange is caused by a reaction between singlet oxygen formed via the dye triplet state and the protein. In order to identify the region where the singlet oxygen reacts with the protein we have investigated the kinetics of the deactivation in presence ofthe inhibitor of the enzymatic reaction N-acetylglucosamine (GlcNAc). The overall experimental rate constant becomes slower with increasing saccharide concentrations. As we can exclude experimentally that this kinetical effect is caused in presence of the saccharide by a physical quenching of singlet oxygen or of the dye triplet state it has to be assumed that GlcNAc protects the surrounding of its bindings place at subsite C of the enzymatic center sterically against an attack of singlet oxygen. In this region three tryptophan residues are located, which could be sensitive against singlet oxygen. Surprisingly, however, it has been found that only those species are protected, in which a second saccharide molecule is bound to the protein, probably at subsite E at the enzymatic center, where no sensitive amino acid side chains are located.
The kinetics of the photodynamic desactivation of lysozyme in presence of acridine orange as the sensitizer have been investigated in detail varying oxygen, protein, dye concentration, ionic strength and pH value. The kinetics can be approximately described as an over all pseudo-first- order rate process. Changing the solvent from water to D2O or by quenching experiments in presence of azide ions it could be shown that the desactivation of lysozyme is caused exclusively by singlet oxygen. The excited oxygen occurs via the triplet state of the dye with a rate constant considerably lower than that to be expected for a diffusionally controlled reaction. Singlet oxygen reacts chemically (desactivation, k=2.9 × 107 ᴍ-1 sec-1) and physically (quenching process, k = 4.1 × 108 ᴍ-1sec-1) with the enzyme. The kinetical analysis shows that additional chemical reactions between singlet oxygen and lysozyme would have only little influence on the kinetics of the desactivation as long as their products would be enzymatically active and their kinetical constants would be less than about 1 × 108 ᴍ-1 sec-1.
Cytochrome c oxidase (COX), the last enzyme of the respiratory chain of aerobic organisms, catalyzes the reduction of molecular oxygen to water. It is a redox-linked proton pump, whose mechanism of proton pumping has been controversially discussed, and the coupling of proton and electron transfer is still not understood. Here, we investigated the kinetics of proton transfer reactions following the injection of a single electron into the fully oxidized enzyme and its transfer to the hemes using time-resolved absorption spectroscopy and pH indicator dyes. By comparison of proton uptake and release kinetics observed for solubilized COX and COX-containing liposomes, we conclude that the 1-μs electron injection into CuA, close to the positive membrane side (P-side) of the enzyme, already results in proton uptake from both the P-side and the N (negative)-side (1.5 H+/COX and 1 H+/COX, respectively). The subsequent 10-μs transfer of the electron to heme a is accompanied by the release of 1 proton from the P-side to the aqueous bulk phase, leaving ∼0.5 H+/COX at this side to electrostatically compensate the charge of the electron. With ∼200 μs, all but 0.4 H+ at the N-side are released to the bulk phase, and the remaining proton is transferred toward the hemes to a so-called “pump site.” Thus, this proton may already be taken up by the enzyme as early as during the first electron transfer to CuA. These results support the idea of a proton-collecting antenna, switched on by electron injection.
The hypothesis of GLIKMAN and ZABRODA (Biochemistry [USSR] 84,, 239 [1969]) that the primary electron donor during photoreduction of manganese(III) in Mn(III)-hydroxychlorin compounds in oxygen free aqueous alkaline solutions is the axially bound OH- ion was tested with Mn(III)-2-a-hydroxyethyl-isochlorin e4. It has been shown that
1) the primary generation of OH radicals upon irradiation of the complex is highly improbable,
2) light is not essential for the reduction reaction,
3) the kinetics of photoreduction of the Mn(III)-compound in 2 N NaOH clearly is not compatible with OH radical formation.